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Accurate translation initiation is essential for efficient cellular production of proteins; missing or mis-selecting the canonical start site can have detrimental consequences. Extensive Saccharomyces cerevisiae research has identified key eukaryotic initiation factors (eIF1, eIF1A, eIF2, eIF3, and eIF5) that govern start codon selection accuracy, but the increasing complexity of higher eukaryotic systems and the associated technical challenges have limited similar research in mammalian cells. Here, we present an in cellulo study of homozygous knock-in mutants in the N-terminal domain of the eIF3c subunit of the multitasking eIF3 complex in human cells. We identified three key amino acid residues whose mutation disrupts eIF3's interaction with eIF5, thereby preventing functional eIF5 binding to pre-initiation complexes. This selectively relaxes start site selection stringency and reduces initiation sensitivity, disrupts the delicate balance of protein levels of the critical regulators eIF5 and eIF1, and alters translation efficiency for nearly 600 proteins. Proteins with N-terminal targeting signals—such as nuclear-encoded mitochondrial proteins—were among those especially affected, resulting in impaired mitochondrial function. These findings establish the first direct causal link between an initiation factor, stringent start site recognition, and impaired mitochondrial respiration and glycolytic activity, implicating the eIF3–eIF5 interaction as a key regulator of energy homeostasis.
